Project Summary As cellular adjuvants of immunity, NKT cells prime and control the maturation of dendritic cells during all immune responses. Therefore, the understanding of the biology of these regulatory cells should lead to their utilization in anti-microbial and cancer vaccines, immunotherapy of chronic infectious diseases, and the prevention of infection in at-risk patients. We have worked for the past two decades on the identification of the natural endogenous ligands of NKT cells thinking that they could be manipulated in vivo. By creating new tools and approaches, we have recently succeeded in demonstrating that ?-glycosylceramides were the ligands of NKT cells. Since publication of our work, and the first submission of this proposal, we have made great progress in understanding this new class of glycolipids for which each metabolic step is to be described. In the thymus, ?Galcer is produced by DCs in the medullary region, whereas ?Glucer is made by cortical thymocytes. To understand the respective roles of each ligand in thymic selection and peripheral activation, we are using CRISPR/Cas9 technology to deconstruct the pathways of synthesis and degradation. Galactose mutarotase (Galm) for ?Galcer and glucose ceramide synthase (UGCG) for ?Glucer are necessary for synthesis; acid ceramidase, acid ? galactosidase, acid ? glucosidase, and catheptsin L are essential for degradation. A powerful new mass spectrometry technique should allow us to measure directly ? anomers from cells and tissues. As the work moves from cell lines to in vivo studies, our labor is divided in three specific aims: Aim 1: Biosynthesis of endogenous ?-galactosylceramides. Following CRISPR/Cas9 gene inactivation, a robust set of phenotypic and functional assays has been implemented to evaluate the role of each molecule in the pathway. The consequences of the elimination of ?Galcer for thymic selection, shaping of the NKT cell repertoire, and peripheral functions will be evaluated in Galm knockout mice. Aim 2: Biosynthesis of endogenous ?- glucosylceramides. The screen of enzymes and transporters involved in the synthesis of glucosylceramides using CRISPR/Cas9 technology has revealed the role of UGCG. Its role as a synthase or the necessity to use an anomerase in a two step synthesis will be explored. The functional consequences of eliminating ?Glucer will be evaluated. Aim 3: Catabolism of ?-linked glycosylceramides. Both ?Glucer and ?Galcer catabolisms are controlled by acid ceramidase, ASAH1, which cleaves off the fatty acid to produce ?-lysoglycosylceramides, a family of potent NKT cell activators. We will evaluate the biological role of these lysolipids by studying their inactivation when ?-linked glucose and galactose are removed by acid ? glycosidases in the lysosome. Cathepsin L functions in processing and activating these various hydrolases will also be examined in cells and animals. The manipulation of degradation of ?Glucer and ?Galcer by using specific enzyme inhibitors will be tested in vivo and evaluated with respect to NKT cell numbers and function in order to establish a new proof of principle for the therapeutic manipulation of NKT cells.